Application Note

Automated slide scanning of histologically stained and fluorescently labelled tissues

  • Locate key regions of interest by rapidly scanning at low magnification
  • Stitch large, overlapping images together automatically
  • Image either histologicallystained or fluorescentlylabelled tissue
  • Set up slide imaging in less than 5 minutes

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Introduction

Although multi-well plate formats for cellbased assays are growing in popularity, there is also an increasing demand for automated imaging of microscope slides. Applications for slide assays include staining and imaging of tissue slices representing various diseased and normal states, performing cell-based assays in array format, and testing assays already developed on slides prior to moving to plate-based formats.

Slides are variable in nature often making them challenging to image on highcontent systems designed for multi-well plates. Unless slides are produced in a predictable format (such as a printed RNAi or antibody array), you typically need to visually determine the specific area(s) for scanning at higher resolutions. Scanning the entire slide with a higher magnification, although desirable, is usually time- and data storage-prohibitive.

The ImageXpress® Screening Systems make selective slide scanning easy, letting you quickly create automated workflows for scanning and high-resolution imaging of regions of interest on either histologicallystained or fluorescently-labelled tissue. Our systems include MetaXpress® High Content Image Acquisition and Analysis Software, which provides user-friendly tools specifically for working with slides.

In this note we describe the Slide Region Acquisition application in MetaXpress Software. It lets you automatically acquire and stitch overlapping images from slides, which is particularly beneficial for tissue slices or other samples that require imaging of large contiguous areas.

Capture low and high magnification quickly using simple workflows

Typical workflows include scanning the entire slide (or multiple slides) first at low magnification. Areas for higherresolution imaging are then selected, either automatically or manually, and imaged to your specifications. Using our automation-friendly three-slide holders, ImageXpress Systems can perform both the low resolution scanning and the highthroughput, high-resolution scanning of specific regions of interest across a library of slides, and do both automatically.

Setting up Slide Region Acquisition
Step Description
1. Load slide Place prepared slide in the slide holder with coverslip facing down. Load into ImageXpress System. Image slides without coverslips as well.
2. Preview slide Go to Slide Scanning > Perform Preview Scan on the MetaXpress Software taskbar. Select a low magnification (1x-4x) and a single wavelength that can be used to view the entire sample with a short exposure, such as Transmitted Light (if available) or DAPI for a nuclear stain. Save time by imaging the entire slide at a single focal position.
3. Define region(s) Draw a region(s) around the area(s) of interest you want scanned at a higher magnification. Go to Slide Scanning > Create Scan Areas to convert regions sites for acquisition.
4. Define scan parameters Select high-resolution imaging parameters including magnification, wavelengths, exposure times, and laser-based or image-based autofocus.
5. Scan Save your settings and start the scan.

Figure 1. Simplified workflow for acquiring slides on ImageXpress Systems.

Set up slide region acquisition in 5 minutes or less

  TMA slide Rat brain slide Kidney slide
Staining H&E DAPI, FITC, TxRed DAPI, FITC, TRITC
# of wavelengths 3 3 3
# of regions 30 1 1
# of sites per region 16 450 200
Total # of images captured 1440 1350 600
Time to set up (low mag and ROI) 5 minutes 3 minutes 3 minutes
Time to acquire (high mag) 30 minutes 41 minutes 19 minutes
Time to analyze 35 minutes *28 minutes NA

*Using MetaXpress PowerCore

Table 1. Slide image acquisition details. Time listed in Time to set up is the only hands-on time needed. Very limited hands-on time (maximum 5 minutes) is required to capture high magnification images of relevant tissues on a slide.

Image acquisition at higher magnification and subsequent analysis is all completely automated, hands-off time. Capture as many as 1440 images in 30 minutes, with the flexibility to do either colorimetric or fluorescent imaging.

Straightforward Tissue Microarray (TMA) acquisition and analysis

Pathological investigation of disease mechanisms occurring at the tissuelevel of organization provide a wealth of knowledge regarding the diseased state. However, acquiring and analyzing images of pathological and normal tissues can be a cumbersome process.

Tissue microarrays were developed to circumvent this time-intensive process. TMA slides are precisely organized tissue sections taken from multiple patients, allowing easy comparison between pathological conditions and normal age-matched controls. They have been extremely useful in the identification of cancerous tissues in combination with histological stains such as H&E. In this section, we describe the acquisition and analysis of an H&E stained TMA slide obtained from Biolabs, Inc.

Lung tissue sections (30 in total: 15 cancerous and 15 age-matched controls) were provided in a TMA format and stained with hematoxylin and eosin to visualize DNA/RNA and proteins, respectively. The TMA slide was scanned with transmitted light and a 4X Plan Apo objective in approximately 2.5 minutes (Figure 2, Top), allowing quick identification of the location of tissue sections on the slide. Next, a region was drawn around each of the 30 tissue sections to define the areas to re-image at higher magnification. Tissue sections were imaged using a 20X ELWD objective, and a 4x4 image grid was collected per sample. Color images were created by illuminating the sample with transmitted light and collecting the light through standard RGB (red, green, and blue) filter cubes.

Figure 2. (A) Preview scan of H&E stained lung tissues using transmitted light and RGB filters. (B–D) Selected regions shown at continually higher resolution. (B) Single tissue image stitched together from 4x4 grid of images captured with a 20X ELWD objective. (D) High resolution image shown with Hematoxylin positive cells identified as blue and eosin positive cells identified as yellow. Analysis was performed in MetaXpress Custom Module Editor software. (E) Layout of TMA slide indicating placement of cancerous and normal lung tissue specimens. (F) Heat map from MetaXpress illustrating the fraction of blue (hematoxylin) positive cells in each respective tissue section. Slide region acquisition on the ImageXpress System along with MetaXpress Software enables accurate identification of cancerous versus normal lung tissue specimens.

Evaluate fluorescently-labeled tissues

Slide scanning has traditionally been performed with colorimetric stains, but many researchers are now moving towards fluorescent markers because of their increased specificity, contrast, diversity, and ease-of-use. Scientists looking for more biologically relevant disease models are also turning to whole tissue imaging. Here, we illustrate how the ImageXpress Systems’ flexibility allows for fluorescence imaging of astrocytes in a slice of rat brain tissue.

Discriminate between neural cell types in a rat brain slice

Here we highlight the image acquisition of a section of brain tissue obtained from a rat. The entire tissue sample was labeled with DAPI, Alexa 488 anti-GFAP, and Alexa 532 anti-F-actin.

The rat brain slide was scanned with transmitted light and a 4X Plan Apo objective in approximately 2.5 minutes (Figure 3, Top) allowing quick identification of the location of the tissue. One region was drawn around the tissue section to define the scan area to cover with the 20X ELWD objective. Fluorescent wavelengths were collected using the standard DAPI, FITC, and TRITC filter cubes (Figure 3).

Figure 3. (A) Low resolution montage of rat brain section allows for quick identification of regions of interest. (B–F) High resolution images (C, E) of fluorescently stained astrocytes were analyzed using the Neurite Outgrowth Application Module in MetaXpress Software. (D, F) Accurate identification of neuronal cell bodies and processes are illustrated by the white mask overlaid on the image.

Identify microstructures within a mouse kidney section

A kidney slide from Molecular Probes was scanned with a DAPI filter and a 4X Plan Apo objective in approximately 2.5 minutes, again allowing quick identification of the location of the tissue. One region was drawn around the tissue section to define the scan area to cover with the 20X ELWD objective. Fluorescent wavelengths were collected using the standard DAPI, FITC, and Texas Red filter cubes to identify various structures within the kidney tissue (Figure 4).

Figure 4. Fluorescent slide of kidney tissue imaged using the Slide Region Acquisition Module in MetaXpress Software. Increasing magnifications shown from regions-of-interest (red outline) allow for seamless transition between whole tissue and higher magnification regions.

Summary

Slide acquisition and analysis is becoming an important tool in translational research and in the high-content screening arena, particularly as investigators attempt to work with more biologically relevant tissue samples. The ImageXpress® High Content Screening Systems coupled with MetaXpress High Content Image Acquisition and Analysis Software guide you through imaging and analysis with simple workflows. Hands-on setup time is only 5 minutes, and both slide-scanning and high-resolution imaging of regions of interest is completely automated-collect as many as 1440 images in just 30 minutes. You’ll also have the flexibility to image and analyze both fluorescently-labeled and histologically-stained tissues. Ready when you are for the ever-evolving future, the platform is also a fully-loaded High Content Analysis system.

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